sequencing riddle...

Daniel Mytelka mytelka at mendel.Berkeley.EDU
Fri Apr 4 19:59:21 EST 1997


Well, one thing you can do is as follows:

1)Find an enzyme that cuts in the sequence

2)Sequence with the known primer. You'll get double bands through
the length of the short fragment, then single bands the rest of the way.

3)Use the clear sequence to synthesize another (unique) primer to
get the rest of the sequence.

Now, technically, the riddle was to use the same primer, and this requires
a new primer (though no cloning).  One other method would be to
sequence with the original primer and make a new primer extended by 
one of the two possible first bases. The resulting primer would have a 3'
mismatch and primer the other strand poorly, giving little or no sequence.
If you wanted to meet the riddle's conditions, it might be possible to
make such a primer from the original primer with terminal deoxynucleotidyl
transferase and a (temporarily) 3' blocked nucleotide...

Daniel Mytelka

In article <199704042300.QAA145216 at nestor.NMSU.Edu>,
Hiranya Roychowdhury <hroychow at NMSU.EDU> wrote:
>Hello All,
>        We have come across an interesting brain teaser. One of our 
colleagues has amplified a piece of DNA from a plant using a single primer 
(i.e. amplified region bordered by inverted repeats...). Now the teaser 
(may not be one for some of you): How can this piece of DNA be sequenced 
WITHOUT having to CLONE it? In other words, the investigator wants to use 
the same primers to sequence the pcr products directly after purifying them! 
This conundrum has occupied parts of our brains for the last week or so. I 
tend to think that this is impossible.... but then there may be some clever 
way which is eluding us...
>
>Humbly,
>
>Hiranya.
>
>Dr. Hiranya Sankar Roychowdhury
>Plant Genetic Engineering Lab.
>New Mexico State University
>Las Cruces, NM 88003
>Ph. (505) 646-5785
>hroychow at nmsu.edu





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